Autosomal recessive congenital cataract linked to EPHA2 in a consanguineous Pakistani family.

Purpose To investigate the genetic basis of autosomal recessive congenital cataracts in a consanguineous Pakistani family. Methods All affected individuals underwent a detailed ophthalmological and clinical examination. Blood samples were collected and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic microsatellite markers. Logarithm of odds (LOD) scores were calculated, and Eph-receptor type-A2 (EPHA2), residing in the critical interval, was sequenced bidirectionally. Results The clinical and ophthalmological examinations suggested that all affected individuals have nuclear cataracts. Genome-wide linkage analyses localized the critical interval to a 20.78 cM (15.08 Mb) interval on chromosome 1p, with a maximum two-point LOD score of 5.21 at θ=0. Sequencing of EPHA2 residing in the critical interval identified a missense mutation: c.2353G>A, which results in an alanine to threonine substitution (p.A785T). Conclusions Here, we report for the first time a missense mutation in EPHA2 associated with autosomal recessive congenital cataracts.

Congenital cataracts are one of the major causes of vision loss in children worldwide and are responsible for about onethird of blindness in infants [1,2]. Congenital cataracts can occur in an isolated fashion or as one component of a syndrome affecting multiple tissues. Nonsyndromic congenital cataracts have an estimated frequency of 1-6 per 10,000 live births [3]. They vary markedly in severity and morphology, affecting the nuclear, cortical, polar, or subcapsular parts of the lens or in severe cases the entire lens.
EPHA2 belongs to the tyrosine kinase family of proteins and is an epithelial cell kinase that has been associated with autosomal dominant cataracts and recently it was implicated in age-related cortical cataracts in humans and mice [17,18]. EPHA2 belongs to the tyrosine kinase family, and EPHA2 is epithelial cell kinase that interacts with membrane-bound ephrin ligands, which play an important role in morphogenesis and in numerous developmental processes [19]. Structurally, these proteins contain a ligand-binding domain, epidermal growth factor-like domain, and tyrosine kinase catalytic domain [20].
Here we report a consanguineous Pakistani family (PKCC118) with four affected members with nuclear cataracts. Genome-wide linkage analyses localized the disease interval to chromosome 1p. Sequencing of EPHA2 identified a missense mutation that segregated with the disease phenotype in the family. To the best of our knowledge this is the first report associating mutations in EPHA2 with autosomal recessive congenital cataracts. 50 μl [10 mg/ml] of proteinase K, 6 ml TNE buffer [10 mM Tris HCl, 2 mM EDTA, 400 mM NaCl] and 200 μl of 10% sodium dodecyl sulfate) was added to the resuspended pellets and incubated overnight in a shaker (250 rpm) at 37 °C. The digested proteins were precipitated by adding 1 ml of 5 M NaCl, followed by vigorous shaking and chilling on ice for 15 min. The precipitated proteins were pelleted by centrifugation at 3,000 rpm for 20 min and removed. The supernatant was mixed with equal volumes of phenol/chloroform/isoamyl alcohol (25:24:1), and the aqueous layer containing the genomic DNA was carefully collected. The DNA was precipitated with isopropanol and pelleted by centrifugation at 4,000 rpm for 15 min. The DNA pellets were washed with 70% ethanol and dissolved in TE buffer. The DNA concentration was determined with a SmartSpec plus Bio-Rad Spectrophotometer (Bio-Rad, Hercules, CA).   Automated Sequencer (Applied Biosystems). Sequencing results were assembled with ABI PRISM sequencing analysis software version 3.7 and analyzed with SeqScape software (Applied Biosystems).

Prediction analysis:
The degree of evolutionary conservation of amino acid at positions of interest and their possible impact on the structure of the EPHA2 protein was examined with SIFT and PolyPhen tools available online. Evolutionary conservation of the mutated amino acid (A785) in other EPHA2 orthologs was examined using the UCSC genome browser.

RESULTS
A large consanguineous family, PKCC118, consisting of four affected and 14 unaffected individuals in three consanguineous marriages, was recruited from the Punjab province of Pakistan ( Figure 1). A detailed medical history was obtained from all the affected and unaffected individuals of the family, which revealed that cataracts in affected Initially, all the known loci and genes for recessive cataract were excluded by haplotype analysis using closely spaced short tandem repeat (STR) markers (data not shown). Subsequently, a genome-wide scan was completed on a subset of family members and LOD scores of 2.36, 3.23, 4.74, and 1.8 at θ=0 were obtained with D1S2697, D1S199, D1S234, and D12S336, respectively. Additional STR markers from the Marshfield database were genotyped for all the participating members of the family, and this excluded the region at chromosome 12 with a LOD score of -∞ for D12S99 and D12S1697 (data not shown). Fine mapping with D1S1592, D1S2826, D1S2644, D1S2864, and D1S2787 yielded LOD scores of 4.56, 2.37, 3.60, 5.21, and 5.08 at θ=0, respectively ( Table 2). Sequencing of EPHA2 identified a missense mutation: c. 2353 G>A that results in a p.A785T, which affects the tyrosine kinase domain of EPHA2. All affected individuals were homozygous for this variation, whereas unaffected individuals were either heterozygous or were homozygous for the wild-type alleles (Figure 3). This variation was not present in 96 ethnically matched control samples. In addition to the pathogenic variation, we identified three single-nucleotide polymorphisms (SNPs) rs2230597, rs3754334, and rs1803527 and a synonymous variation: c.453 C>T (p.T151T).
To investigate the possible impact of the p.A785T substitution on the EPHA2 protein, we used SIFT and PolyPhen software. SIFT predictions suggested that A785T substitution will not be tolerated by the native threedimensional structure of the EPHA2 protein. The affected protein function score for A785T was 0.01(amino acids with probabilities <0.05 are predicted to be deleterious). Likewise, position-specific score differences obtained from PolyPhen suggested that A785T substitution could potentially have a deleterious effect on the EPHA2 protein structure. The position-specific independent counts (PSIC) score difference was 2.09 (a PSIC score difference >1.0 is probably damaging). We further analyzed the evolutionary conservation of the substituted amino acids by aligning EPHA2 orthologues. The results strongly suggest that not only Ala785 but also amino acids in the immediate neighborhood are well conserved among EPHA2 orthologues ( Figure 4).

DISCUSSION
Here, we report mutations in EPHA2 associated with autosomal recessive congenital cataracts in a consanguineous Pakistani family. Genome-wide scan localized the critical interval to chromosome 1p with maximum two-point LOD scores of 5.21 at θ=0. Sequencing of EPHA2 identified a missense mutation that segregates with the disease phenotype in the family. This mutation is predicted to have a deleterious effect on the native structure of the protein and was absent in 96 ethnically matched control samples. Taken together, these results strongly suggest that mutation in EPHA2 is responsible for recessive congenital cataracts in the Pakistani family.
The linked interval on chromosome 1p36.21-p35.2 was previously mapped in American, British, and Australian families with dominant congenital cataract in three independent reports [24][25][26]. Recently, Shiels and colleagues [17] reported mutations in EPHA2 that are responsible for autosomal dominant cataracts. During the preparation of this article, Zhang and colleagues [27] identified mutations in a Chinese and two previously reported British and Australian kindreds, confirming the involvement of EPHA2 in autosomal dominant cataract.
The human lens is an avascular tissue, where cell-cell junctions are critical for providing nutrient transport and removal of metabolic wastes through intercellular adhesion complexes comprising gap and adherens junctions. Adherens junctions of lens are provided with transmembrane proteins and N-cadherin and β-catenin proteins [28]. Recently, it was demonstrated that ephrin-A5 acts as a regulator for EPHA2, and loss of ephrin-A5 function can lead to cataracts in mice The asterisk indicates marker included in genome wide scan. [29]. Further, it has been shown that ephrin-A5 interacts with the EphA2 receptor to regulate the adherens junction complex by enhancing recruitment of β-catenin to N-cadherin [29]. Transparency and precise shape are distinctive features of the lens that are critical for proper light refraction; however, not much is known about the mechanisms that maintain transparency of lens. Elucidating the molecular mechanisms and factors that maintain or disrupt lens transparency is a fundamental precursor for preventing cataract. Identification of new genes associated with cataractogensis will help us better understand the molecular biology of the human lens, including structural and metabolic mechanisms involved in maintaining the clarity of the lens.